It has become common practice in recent years for chromatographers to form cross-linked polymer stationary phases within capillary columns in order to improve the performance of capillary gas chromatography within the columns. Because splitless and on-column injection techniques both flood the initial length of a column with solvent, which is sometimes at elevated temperatures, an untreated column will suffer progressive phase loss and resultant diminished efficiency due to rearrangement of the stationary-phase film. Alternatively, a properly cross-linked capillary column can be subjected to repeated solvent exposure without affecting its solute retention characteristics.
A further benefit arising from the insolubility of cross-linked polymer phases in such columns is the freedom to wash the columns with solvent for the purpose of removing non-volatile sample components. If these compounds fail to elute during a normal analysis, background noise can increase and interactions may occur with other sample components during subsequent analyses. The ability of cross-linked phases to resist dissolution has also facilitated the development of capillary supercritical fluid chromatography and open-tubular liquid chromatography with partition retention mechanisms.
Yet another advantage of cross-linking polymer coatings within such columns is to enhance the film stability. The relationship of the stationary-phase surface tension to the free surface energy of the columns substrate determines whether film disruption is thermodynamically favored. The kinetics of droplet formation however are greatly influenced by polymer viscosity. Thus, polar polysiloxane phases, which are normally subject to film disruption at elevated temperatures due to reductions in phase viscosity, will have enhanced physical stability when cross-linked.
There are known in the prior art a variety of techniques for cross-linking polymer phases in chromatographic columns. Siloxane polymers have received the most attention in this regard, but other phase classes have also been successfully cross-linked. Two distinctly different types of cross-linking can be considered for such applications, i.e., those with linkages formed either through the linear backbone of the polymer, or those with the linkages formed through substituent groups.
It is also known that in situ concatenation of a partially polymerized siloxane results in highly stable chromatographic column coatings. Cross-linked phases have been successfully prepared by adding tri- or tetra-functional silanes during the polymerization step to form the desired cross-linked phases. However, a major disadvantage of both of those known approaches is that they invariably suffer from increased stationary-phase activity due to the presence of uncapped functional sites on the silicon atoms.
A major improvement over such earlier known techniques was the application of free radical induced cross-linking to gas chromatography stationary phases. That approach, which was directly adapted from basic silicone chemistry developed in the 1950's, created cross-linkages through substituent groups, while leaving the polymer backbone intact. Because polymers that are not based on the siloxanes can have the same substituents, free radical cross-linking has also been applied to a broader range of stationary phases including polyethylene glycols. An advantage of this method is that it can be performed in situ after the phase has been deposited on a column wall.
Free radical cross-linking involves a chain reaction that is stimulated by a free radical initiator. Several different initiators have been successfully used. Earlier experiments were with organic peroxides, which decomposed upon heating to yield free radicals. The incorporation of active by-products into the stationary-phase layer was a problem with organic peroxides, as evidenced by adsorption of polar solutes. A second drawback was the elevated temperatures required to cause free radical formation. When heated, those phases exhibiting only marginal physical stability may coalesce into droplets, thus drastically reducing the operating efficiency of the column.
With each of the various chemical cross-linking agents, the incorporation of residual groups into the polymer structure can cause residual activity. A further drawback is that chemical initiators, except for ozone, which reacts spontaneously at room temperature, must be heated before cross-linking occurs. Even though the temperatures used to stimulate cross-linking are usually well below those encountered during separations, it may be preferable to avoid heating some polar phases before their mechanical stability has been augmented by cross-linking.
It has also been reported that free radicals can be formed directly in a stationary phase by irradiation with gamma rays. However columns prepared in this manner still exhibit undesirable adsorption of polar solutes. Other problems with irradiative cross-linking include damage to the outer polyimide coating and the general inaccessibility of suitable facilities for accomplishing such irradiation.